Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants.

Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position 71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1306 are likely benign. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.

Selection on synonymous sites: the unwanted transcript hypothesis.

Although translational selection to favour codons that match the most abundant tRNAs is not readily observed in humans, there is nonetheless selection in humans on synonymous mutations. We hypothesize that much of this synonymous site selection can be explained in terms of protection against unwanted RNAs - spurious transcripts, mis-spliced forms or RNAs derived from transposable elements or viruses. We propose not only that selection on synonymous sites functions to reduce the rate of creation of unwanted transcripts (for example, through selection on exonic splice enhancers and cryptic splice sites) but also that high-GC content (but low-CpG content), together with intron presence and position, is both particular to functional native mRNAs and used to recognize transcripts as native. In support of this hypothesis, transcription, nuclear export, liquid phase condensation and RNA degradation have all recently been shown to promote GC-rich transcripts and suppress AU/CpG-rich ones. With such 'traps' being set against AU/CpG-rich transcripts, the codon usage of native genes has, in turn, evolved to avoid such suppression. That parallel filters against AU/CpG-rich transcripts also affect the endosomal import of RNAs further supports the unwanted transcript hypothesis of synonymous site selection and explains the similar design rules that have enabled the successful use of transgenes and RNA vaccines.

The RNA-Binding Landscape of HAX1 Protein Indicates Its Involvement in Translation and Ribosome Assembly.

HAX1 is a human protein with no known homologues or structural domains. Mutations in the HAX1 gene cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported the RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here, we report the transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in translation, ribosome biogenesis, and rRNA processing. Using CRISPR knockouts, we find that HAX1 RNA targets partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. The functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.

Lighting up protein design.

Using a neural network to predict how green fluorescent proteins respond to genetic mutations illuminates properties that could help design new proteins.

Global mapping of RNA homodimers in living cells.

RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding RNA dimerization has been hampered by the lack of systematic in vivo detection methods. Here, we show that CLASH, PARIS, and other RNA proximity ligation methods detect RNA homodimers transcriptome-wide as "overlapping" chimeric reads that contain more than one copy of the same sequence. Analyzing published proximity ligation data sets, we show that RNA:RNA homodimers mediated by direct base-pairing are rare across the human transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA, and a subset of tRNAs. Mutations in the homodimerization domain of U8 snoRNA impede dimerization in vitro and disrupt zebrafish development in vivo, suggesting an evolutionarily conserved role of this domain. Analysis of virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N gene in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies.

In vivo structure and dynamics of the SARS-CoV-2 RNA genome.

The dynamics of SARS-CoV-2 RNA structure and their functional relevance are largely unknown. Here we develop a simplified SPLASH assay and comprehensively map the in vivo RNA-RNA interactome of SARS-CoV-2 genome across viral life cycle. We report canonical and alternative structures including 5'-UTR and 3'-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in both cells and virions. We provide direct evidence of interactions between Transcription Regulating Sequences, which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE. More importantly, we find that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome domains remain stable but with strengthened demarcation of local domains and weakened global cyclization. Taken together, our analysis reveals the structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, the alternative conformations and the maintenance of global genome organization during the whole life cycle of SARS-CoV-2, which we anticipate will help develop better antiviral strategies.

Evidence in disease and non-disease contexts that nonsense mutations cause altered splicing via motif disruption.

Transcripts containing premature termination codons (PTCs) can be subject to nonsense-associated alternative splicing (NAS). Two models have been evoked to explain this, scanning and splice motif disruption. The latter postulates that exonic cis motifs, such as exonic splice enhancers (ESEs), are disrupted by nonsense mutations. We employ genome-wide transcriptomic and k-mer enrichment methods to scrutinize this model. First, we show that ESEs are prone to disruptive nonsense mutations owing to their purine richness and paucity of TGA, TAA and TAG. The motif model correctly predicts that NAS rates should be low (we estimate 5-30%) and approximately in line with estimates for the rate at which random point mutations disrupt splicing (8-20%). Further, we find that, as expected, NAS-associated PTCs are predictable from nucleotide-based machine learning approaches to predict splice disruption and, at least for pathogenic variants, are enriched in ESEs. Finally, we find that both in and out of frame mutations to TAA, TGA or TAG are associated with exon skipping. While a higher relative frequency of such skip-inducing mutations in-frame than out of frame lends some credence to the scanning model, these results reinforce the importance of considering splice motif modulation to understand the etiology of PTC-associated disease.

Causes and Consequences of Purifying Selection on SARS-CoV-2.

Owing to a lag between a deleterious mutation's appearance and its selective removal, gold-standard methods for mutation rate estimation assume no meaningful loss of mutations between parents and offspring. Indeed, from analysis of closely related lineages, in SARS-CoV-2, the Ka/Ks ratio was previously estimated as 1.008, suggesting no within-host selection. By contrast, we find a higher number of observed SNPs at 4-fold degenerate sites than elsewhere and, allowing for the virus's complex mutational and compositional biases, estimate that the mutation rate is at least 49-67% higher than would be estimated based on the rate of appearance of variants in sampled genomes. Given the high Ka/Ks one might assume that the majority of such intrahost selection is the purging of nonsense mutations. However, we estimate that selection against nonsense mutations accounts for only ∼10% of all the "missing" mutations. Instead, classical protein-level selective filters (against chemically disparate amino acids and those predicted to disrupt protein functionality) account for many missing mutations. It is less obvious why for an intracellular parasite, amino acid cost parameters, notably amino acid decay rate, is also significant. Perhaps most surprisingly, we also find evidence for real-time selection against synonymous mutations that move codon usage away from that of humans. We conclude that there is common intrahost selection on SARS-CoV-2 that acts on nonsense, missense, and possibly synonymous mutations. This has implications for methods of mutation rate estimation, for determining times to common ancestry and the potential for intrahost evolution including vaccine escape.

Transcription, mRNA Export, and Immune Evasion Shape the Codon Usage of Viruses.

The nucleotide composition, dinucleotide composition, and codon usage of many viruses differ from their hosts. These differences arise because viruses are subject to unique mutation and selection pressures that do not apply to host genomes; however, the molecular mechanisms that underlie these evolutionary forces are unclear. Here, we analyzed the patterns of codon usage in 1,520 vertebrate-infecting viruses, focusing on parameters known to be under selection and associated with gene regulation. We find that GC content, dinucleotide content, and splicing and m6A modification-related sequence motifs are associated with the type of genetic material (DNA or RNA), strandedness, and replication compartment of viruses. In an experimental follow-up, we find that the effects of GC content on gene expression depend on whether the genetic material is delivered to the cell as DNA or mRNA, whether it is transcribed by endogenous or exogenous RNA polymerase, and whether transcription takes place in the nucleus or cytoplasm. Our results suggest that viral codon usage cannot be explained by a simple adaptation to the codon usage of the host-instead, it reflects the combination of multiple selective and mutational pressures, including the need for efficient transcription, export, and immune evasion.

Evidence for Strong Mutation Bias toward, and Selection against, U Content in SARS-CoV-2: Implications for Vaccine Design.

Large-scale re-engineering of synonymous sites is a promising strategy to generate vaccines either through synthesis of attenuated viruses or via codon-optimized genes in DNA vaccines. Attenuation typically relies on deoptimization of codon pairs and maximization of CpG dinucleotide frequencies. So as to formulate evolutionarily informed attenuation strategies that aim to force nucleotide usage against the direction favored by selection, here, we examine available whole-genome sequences of SARS-CoV-2 to infer patterns of mutation and selection on synonymous sites. Analysis of mutational profiles indicates a strong mutation bias toward U. In turn, analysis of observed synonymous site composition implicates selection against U. Accounting for dinucleotide effects reinforces this conclusion, observed UU content being a quarter of that expected under neutrality. Possible mechanisms of selection against U mutations include selection for higher expression, for high mRNA stability or lower immunogenicity of viral genes. Consistent with gene-specific selection against CpG dinucleotides, we observe systematic differences of CpG content between SARS-CoV-2 genes. We propose an evolutionarily informed approach to attenuation that, unusually, seeks to increase usage of the already most common synonymous codons. Comparable analysis of H1N1 and Ebola finds that GC3 deviated from neutral equilibrium is not a universal feature, cautioning against generalization of results.

RNA Conformation Capture by Proximity Ligation.

RNA proximity ligation is a set of molecular biology techniques used to analyze the conformations and spatial proximity of RNA molecules within cells. A typical experiment starts with cross-linking of a biological sample using UV light or psoralen, followed by partial fragmentation of RNA, RNA-RNA ligation, library preparation, and high-throughput sequencing. In the past decade, proximity ligation has been used to study structures of individual RNAs, networks of interactions between small RNAs and their targets, and whole RNA-RNA interactomes, in models ranging from bacteria to animal tissues and whole animals. Here, we provide an overview of the field, highlight the main findings, review the recent experimental and computational developments, and provide troubleshooting advice for new users. In the final section, we draw parallels between DNA and RNA proximity ligation and speculate on possible future research directions.

Codon Usage and Splicing Jointly Influence mRNA Localization.

In the human genome, most genes undergo splicing, and patterns of codon usage are splicing dependent: guanine and cytosine (GC) content is the highest within single-exon genes and within first exons of multi-exon genes. However, the effects of codon usage on gene expression are typically characterized in unspliced model genes. Here, we measured the effects of splicing on expression in a panel of synonymous reporter genes that varied in nucleotide composition. We found that high GC content increased protein yield, mRNA yield, cytoplasmic mRNA localization, and translation of unspliced reporters. Splicing did not affect the expression of GC-rich variants. However, splicing promoted the expression of AT-rich variants by increasing their steady-state protein and mRNA levels, in part through promoting cytoplasmic localization of mRNA. We propose that splicing promotes the nuclear export of AU-rich mRNAs and that codon- and splicing-dependent effects on expression are under evolutionary pressure in the human genome.

COMRADES determines in vivo RNA structures and interactions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

Codon usage influences fitness through RNA toxicity.

Many organisms are subject to selective pressure that gives rise to unequal usage of synonymous codons, known as codon bias. To experimentally dissect the mechanisms of selection on synonymous sites, we expressed several hundred synonymous variants of the GFP gene in Escherichia coli, and used quantitative growth and viability assays to estimate bacterial fitness. Unexpectedly, we found many synonymous variants whose expression was toxic to E. coli Unlike previously studied effects of synonymous mutations, the effect that we discovered is independent of translation, but it depends on the production of toxic mRNA molecules. We identified RNA sequence determinants of toxicity and evolved suppressor strains that can tolerate the expression of toxic GFP variants. Genome sequencing of these suppressor strains revealed a cluster of promoter mutations that prevented toxicity by reducing mRNA levels. We conclude that translation-independent RNA toxicity is a previously unrecognized obstacle in bacterial gene expression.

RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit.

Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In particular, pre-mRNA splicing was reported to be associated with slowed RNAPII elongation. Here, we identify a site of ubiquitination (K1246) in the catalytic subunit of RNAPII close to the DNA entry path. Ubiquitination was increased in the absence of the Bre5-Ubp3 ubiquitin protease complex. Bre5 binds RNA in vivo, with a preference for exon 2 regions of intron-containing pre-mRNAs and poly(A) proximal sites. Ubiquitinated RNAPII showed similar enrichment. The absence of Bre5 led to impaired splicing and defects in RNAPII elongation in vivo on a splicing reporter construct. Strains expressing RNAPII with a K1246R mutation showed reduced co-transcriptional splicing. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.

N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3'-end processing.

N6-methyladenosine (m6A) is the most abundant base modification found in messenger RNAs (mRNAs). The discovery of FTO as the first m6A mRNA demethylase established the concept of reversible RNA modification. Here, we present a comprehensive transcriptome-wide analysis of RNA demethylation and uncover FTO as a potent regulator of nuclear mRNA processing events such as alternative splicing and 3΄ end mRNA processing. We show that FTO binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced (AS) exons and poly(A) sites. FTO knockout (KO) results in substantial changes in pre-mRNA splicing with prevalence of exon skipping events. The alternative splicing effects of FTO KO anti-correlate with METTL3 knockdown suggesting the involvement of m6A. Besides, deletion of intronic region that contains m6A-linked DRACH motifs partially rescues the FTO KO phenotype in a reporter system. All together, we demonstrate that the splicing effects of FTO are dependent on the catalytic activity in vivo and are mediated by m6A. Our results reveal for the first time the dynamic connection between FTO RNA binding and demethylation activity that influences several mRNA processing events.

Mutually Exclusive CBC-Containing Complexes Contribute to RNA Fate.

The nuclear cap-binding complex (CBC) stimulates processing reactions of capped RNAs, including their splicing, 3'-end formation, degradation, and transport. CBC effects are particular for individual RNA families, but how such selectivity is achieved remains elusive. Here, we analyze three main CBC partners known to impact different RNA species. ARS2 stimulates 3'-end formation/transcription termination of several transcript types, ZC3H18 stimulates degradation of a diverse set of RNAs, and PHAX functions in pre-small nuclear RNA/small nucleolar RNA (pre-snRNA/snoRNA) transport. Surprisingly, these proteins all bind capped RNAs without strong preferences for given transcripts, and their steady-state binding correlates poorly with their function. Despite this, PHAX and ZC3H18 compete for CBC binding and we demonstrate that this competitive binding is functionally relevant. We further show that CBC-containing complexes are short lived in vivo, and we therefore suggest that RNA fate involves the transient formation of mutually exclusive CBC complexes, which may only be consequential at particular checkpoints during RNA biogenesis.

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E.

RNA sequencing studies have identified hundreds of non-coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high-throughput analysis of RNA-RNA interactions in bacteria. Here we demonstrate that in vivo sRNA-mRNA duplexes can be recovered using UV-crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base-paired sRNA-mRNA duplexes in association with RNase E, allowing proximity-dependent ligation and sequencing of cognate sRNA-mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA-mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co-regulated target mRNAs. We identified multiple mRNA targets for the pathotype-specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli Numerous sRNA interactions were also identified with non-coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.